The long-term goal of the proposed research is to understand how DNA replication is regulated such that a precise copy of the genome is made. Accurate replication of the genome and continuous surveillance of its integrity are essential for cell survival and the avoidance of diseases such as cancer and premature aging. The genome is constantly exposed to environmental and endogenous genotoxic insults that challenge DNA replication. The replication stress response (RSR) is a subset of the DNA damage response (DDR) that acts during every cell division cycle to deal with these challenges and promote the faithful duplication of the genome. One component of the replication stress response pathway is the checkpoint kinase ATR. ATR promotes faithful DNA replication by regulating origin firing and cell cycle progression, stabilizing stalled replication forks, and promoting the repair and recovery of stalled forks. The mechanisms mediating many of these ATR-regulated activities are poorly understood. We have completed a series of genetic and biochemical screens to define the mechanisms by which the replication stress response regulates DNA replication and maintains genetic stability. These screens included two-hybrid and mass-spectrometry screens to identify proteins that interact with the ATR-ATRIP complex and the TopBP1 regulator of ATR; RNAi screens to define genes that are important for cells to recover from replication fork stress; and RNAi and cDNA overexpression screens to identify genes that when deregulated cause genetic instability. By focusing on proteins identified in more than one of these biochemical and genetic screens and using published genomic/proteomic information, we identified thirty-six high confidence RSR proteins. These include twenty-four candidate RSR proteins not previously linked to replication stress responses. We hypothesize that these novel RSR proteins have distinct functions in replication and DNA damage responses. This hypothesis will be tested using a combination of genetic and biochemical approaches. Completion of this research proposal will define the genome maintenance activities of novel replication stress response proteins and improve our understanding of how a precise copy of the genome is made during every cell division cycle.